Polymerase Chain Reaction- Part II: Validation, Optimization, Limitations and Applications

Polymerase Chain Reaction (PCR) is an in vitro technique based on the principle of DNA polymerization reaction by which a particular DNA sequence can be amplified and made into multiple copies. Using this technique scientists have now been able to study genes and proteins in a much better way and this technique has boosted the field of biotechnology the world over. This article is split into three parts and covers the following topics:

  1. Part I: Principle, Components, Procedure and Stages of PCR
  2. Part II: Validation, Optimization, Limitations and Applications (Current Article)
  3. Part III: Variations or Types of PCR and Future prospects of PCR

Part II

Validation of Polymerase Chain Reaction

Polymerase chain reaction can be validated by running a gel electrophoresis. The PCR product is pipetted into a special agar that will separate the DNA fragments according to their weight by using electricity. After running the gel the fragments can be seen with a UV light. The reason we can see the fragments with UV light is due to the Ethidium bromide dye that has been added to the gel. This dye will adhere to the DNA in the gel and when the gel is finished it can be viewed under a UV light and can be photographed.

Optimization of Polymerase Chain Reaction

PCR has several characteristics such as specificity, efficiency (yield of product), sensitivity and error rate. By measuring these qualitative characters we can know how accurately the PCR works. [9]

1. Sensitivity of PCR one can increased by

  • Increasing concentration of primers (0.2-1 uM final concentration).
  • Increasing concentration of Taq DNA polymerase (0.25-0.5 units).
  • Increasing number of cycles.
  • Increasing /decreasing annealing temperature.
  • Increasing /decreasing annealing and extending time.

2. Specificity of PCR one can increase by

  • Decreasing concentration of Taq DNA polymerase.
  • Reducing annealing time.
  • Reducing extending time.
  • Rncreasing annealing temperature.
  • Decreasing number of cycles.
  • Using “Hot Start” technique.

3. Yield of PCR products is improved by increasing

  • dNTP concentration (0.1-0.5 mM final concentration).
  • Mg2+ concentration (1-5 mM final concentration).
  • Taq DNA polymerase concentration.
  • Annealing time.
  • Extending time.
  • Number of cycles.

4. Fidelity of PCR one can increase by

  • Decreasing dNTP concentration.
  • Decreasing Taq DNA polymerase concentration.
  • Decreasing Mg2+ concentration.
  • Minimizing reaction time at high temperatures.
  • Decreasing number of cycles.
  • Lowering pH in a reaction mixture.

Limitations of Polymerase Chain Reaction

While a very powerful technique, PCR can also be very tricky. The polymerase reaction is very sensitive to the levels of divalent cations (especially Mg2+) and nucleotides, and the conditions for each particular application must be worked out. Primer design is extremely important for effective amplification. The primers for the reaction must be very specific for the template to be amplified. Cross reactivity with non-target DNA sequences results in non-specific amplification of DNA. Also, the primers must not be capable of annealing to themselves or each other, as this will result in the very efficient amplification of short nonsense DNAs.

The reaction is limited in the size of the DNAs to be amplified (i.e. the distance apart that the primers can be placed). The most efficient amplification is in the 300-1000 bp range. However amplification of products up to 4 Kb has been reported. Also, Taq polymerase has been reported to make frequent mismatch mistakes when incorporating new bases into a strand. The most important consideration in PCR is contamination. If the sample that is being tested has even the smallest contamination with DNA from the target, the reaction could amplify this DNA and report a falsely positive identification.

Application of PCR in Genetics and Biotechnology

Polymerase chain reaction developed by Kary Mullis has been a very useful tool in molecular genetics. It is sometimes referred to as “molecular photocopying” PCR thus can characterize, analyze and synthesize any specific piece of DNA or RNA. With the improvement of PCR protocols and due to the availability of automatic thermocyclers commercially, the application of PCR have increased to a very great extent. Some applications of PCR are as follows:

1. Polymerase chain reaction and archeology.

Amplification and analyzing DNA from bones and mummified soft tissues scientists have been able to correctly identify human origin and races. They have been able to decode the genetic information of extinct species of animals, birds and plants from their fossils.

2. Study of DNA polymorphism using polymerase chain reaction.

Using PCR polymorphism can be studied at loci with known DNA sequence. Sequences of prolamin gene and phytochrome gene have been used for the study of polymorphism for these genes in different species rice plant. [10] Thus they have been able to find the relationship between wild type species and its mutants. It can be used to study DNA polymorphism in the genome using sequences known as primers. With the help of RAPD (Random amplified polymeric DNA) and RAPD maps have been constructed for maize, soya bean, mouse, man etc. [13]

3. PCR and microbiology.

The method is useful for coding the genetic sequence of various disease causing bacteria, fungi, and viruses as well as also harmless species. It can, for example, detect the AIDS virus sooner during the first few weeks after infection than the standard ELISA test. Thus it is useful for the identification, characterization of disease causing organism. [13]

4. Transgene detection.

It can be used to detect the presence of gene transferred into an organism by using the end sequence of transgene for amplification of DNA. The amplified DNA is detected as a band on the electrophoretic gel. Southern blotting, dot blot, northern hybridization can also do the detection, PCR is quicker and faster and it does not use radioactive elements. In gene therapy experiments, the transfer and presence of a marker gene for neomycin resistance (NeoR) could be detected in the blood of patients even after 60 days. It is simple but very efficient. [10][13]

5. Classification of organism.

Gene with greatest polymorphism generally produce largest amount of data for classification of new species. By using the polymerase chain reaction technique amplifications of conserved portions of gene from various organisms can be done and thus classification of organism becomes lucid. The DNA sequence of the PCR amplification can be used to determine taxonomic relationships and build an evolutionary tree.

6. Polymerase chain reaction and drug discovery.

One example is the development and validation of genes encoding drug targets in Streptococcus pneumoniae. It involves the generation of amplicons from Streptococcus pneumoniae DNA that correspondes to known chromosomal regions. These amplicons are organized into pools. The PCR amplicons generated under error-prone conditions generate random mutations into the DNA. Since some of the mutations occur in drug target-encoding genes and subsequently affect the binding of the drug to its respective cellular target, amplicons containing drug targets can be identified as those producing drug-resistant colonies when transformed into Streptococcus pneumoniae.  The binding site of receptor protein can be amplified and studied thoroughly to generate specific drug. [12]

7. PCR and embryology.

DNA amplification of individual sperms is used to determine the frequency of recombination between specified genes. The recompination frequencies can be used to construct linkage maps. PCR amplification can be used to determine the physical location of genes in chromosomes. It is also used to determine sex of embryos. [13]

8. Molecular mapping using polymerase chain reaction:

Genetic and chromosome maps of plants and animals have been generated using PCR. Complete molecular maps of several human chromosomes could be prepared using STS’s and PCR. [10]

9. Gene tagging using polymerase chain reaction.

Molecular markers closely linked to specific gene can be developed using PCR. In tomato, 144 random primers were used to produce 625 PCR products from a set of near isogenic lines for Pseudomonas resistance. At least three of these PCR products were present in one and not in the other in a pair if NIR’s showing linkage with Pseudomonas resistance gene. [10]

10. Prenatal diagnosis using polymerase chain reaction.

Prenatal diagnosis of various diseases like beta-thalassemia, hemophilia, sickle cell anemia etc. can be diagnosed using PCR. [13]

11 . DNA fingerprinting using PCR.

DNA fingerprinting done with the help of PCR helps in the identification of criminals and disputed parentage. PCR allows amplification of DNA from individual hairs, stain of blood or seminal fluid are subjected to southern blotting and DNA hybridization with the help of PCR generated probes. These probes correspond to minisatellites in DNA which are scattered in large number throughout the genome. This identifies the polymorphism in DNA, which is very stable inheritance. [13]

Books on Polymerase Chain Reaction (PCR)

Check out these books on PCR (polymerase chain reaction)


  1. Mullis, Kary (1990). “The unusual origin of the polymerase chain reaction”. Scientific American 262 (4): 56-61, 64-65
  2. “PCR Primer Design Guidelines” (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html)
  3. Rychlik W, Spencer WJ, Rhoads RE (1990). “Optimization of the annealing temperature for DNA amplification in vitro”. Nucl Acids Res 18 (21): 6409–6412
  4. Sharkey, D. J., Scalice, E. R., Christy, K. G., Atwood, S. M., Daiss, J. L. (1994). “Antibodies as Thermolabile Switches: High Temperature Triggering for the Polymerase Chain Reaction”. Bio/Technology 12 (5): 506–509.
  5. “Polymerase chain reaction” (http://en.wikipedia.org/wiki/Polymerase_chain_reaction)
  6. Chien A, Edgar DB, Trela JM (1976). “Deoxyribonucleic acid polymerase from the extreme thermophileThermus aquaticus” J. Bacteriol 174 (3): 1550-1557
  7. Lawyer, F., Stoffel, S., Saiki, R., Chang, S., Landre, P., Abramson, R., Gelfand, D. (1993). “High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity”. PCR methods and applications 2 (4): 275-287
  8. Doris M. Kuehnelt, Elisabeth Kukovetz, Herwig P. Hofer, and Rudolf J. Schaur. 1994. “Quantitative PCR of Bacteriophage lambda DNA Using a Second-Generation Thermocycler” Genome Res. 1994 3: 369-37
  9. G. Zangenberg, R. K. Saiki, and R. Reynolds. “MULTIPLEX PCR: OPTIMIZATION GUIDELINES”
  10. Gupta PK. 1999.”Polymerase chain reaction (PCR) and Gene Amplification” Pp.70-83 in ELEMENTS OF BIOTECHNOLOGY. 1st ed. Rastogi Publications
  11. Norman Arnheim, Tom White, and William E. Rainey. 1990. “The virtually unlimited uses of PCR in evolutionary biology, zoology, botany, animal behavior, conservation biology, environmental science, and ecology” BioScience 4:174-182, March 1990
  12. Aimee E. Belanger, Angel Lai, Marcia A. Brackman, and Donald J. LeBlanc. 2002. “PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for Streptococcus pneumoniae” ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2002, p. 2507-2512
  13. SINGH BD.1998.”Recombinant DNA technology” Pp. 12-93 in BIOTECHNOLOGY.1st ed. Kalyani Publications
  14. Singh OP, Goswami Geeta, Nanda N, Raghavendra K, Chandra D,  and Subbarao SK. 2004. “An allele-specific polymerase chain reaction assay for the differentiation of members of the Anopheles culicifaciescomplex.”
  15. Claude Pirmez, Vale Ria Da Silva Trajano, Manoel Paes-Oliveira Neto, Alda Maria Da-Cruz, Sylvio Celso Gonc¸Alves-Da-Costa, Marcos Catanho, Wim Degrave, and Octavio Fernandes: 1999. “Use of PCR in Diagnosis of Human American Tegumentary Leishmaniasis in Rio de Janeiro, Brazil” J Clin Microbiol. 1999 Jun; 37(6):1819-23
  16. Carsten Goessl et al. “Detection of prostate cancer using methylation-specific PCR”
  17. Athale UH, Shurtleff SA, Jenkins JJ, Poquette CA, Tan M, Downing JR, Pappo AS. “Use of reverse transcriptase polymerase chain reaction for diagnosis and staging of alveolar rhabdomyosarcoma, Ewing sarcoma family of tumors, and desmoplastic small round cell tumor. 2001.” J Pediatr Hematol Oncol. 2001 Feb;23(2):99-104
  18. Yin W, Wang X, Ding Y, Peng H, Liu YL, Wang RG, Yang YL, Xiong JH, Kang SX. 2011. “Expression of Nuclear Factor -κBp65 in Mononuclear Cells in Kawasaki Disease and its Relation to Coronary Artery Lesions.” Indian J Pediatr. 2011 Jun 18. [Epub ahead of print]
  19. Brantsaeter AB, Holberg-Petersen M, Jeansson S, Goplen AK, Bruun JN.2007. “CMV quantitative PCR in the diagnosis of CMV disease in patients with HIV-infection – a retrospective autopsy based study.” BMC Infect Dis. 2007 Nov 6;7:127
  20. Du WD, Chen G, Cao HM, Jin QH, Liao RF, He XC, Chen DB, Huang SR, Zhao H, Lv YM, Tang HY, Tang XF, Wang YQ, Sun S, Zhao JL, Zhang XJ.2011. “Du WD, Chen G, Cao HM, Jin QH, Liao RF, He XC, Chen DB, Huang SR, Zhao H, Lv YM, Tang HY, Tang XF, Wang YQ, Sun S, Zhao JL, Zhang XJ.” Dis Markers. 2011 Jan 1;30(4):181-90
  21. Ramprasath T, Senthil Murugan P, Prabakaran AD, Gomathi P, Rathinavel A, Selvam GS.2011. “Potential risk modifications of GSTT1, GSTM1 and GSTP1 (glutathione-S-transferases) variants and their association to CAD in patients with type-2 diabetes” Biochem Biophys Res Commun. 2011 Apr 1; 407(1): 49-53. Epub 2011 Feb 23
  22. Rapeah Suppian, Zainul Fadziruddin Zainuddin, Mohd Nor Norazmi. 2006. “CLONING AND EXPRESSION OF MALARIA AND TUBERCULOSIS EPITOPES IN MYCOBACTERIUM BOVIS BACILLE CALMETTE-GUERIN” Malaysian Journal of Medical Sciences, Vol. 13, No. 1, January 2006:13-20
  23. Chow WH, McCloskey C, Tong Y, Hu L, You Q, Kelly CP, Kong H, Tang YW, Tang W. 2008. “Application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic Clostridium difficile.” J Mol Diagn. 2008 Sep;10(5):452-8. Epub 2008 Jul 31
  24. Engelstad H, Carney G, Saulis D, Rise J, Sanger WG, Rudd MK, Richard G, Carr CW, Abdul-Rahman OA, Rizzo WB. 2011. “Large contiguous gene deletions in Sjogren-Larsson syndrome” Mol Genet Metab. 2011 May 30. [Epub ahead of print]
  25. Ravi Kumar A, Sathish V, Balakrish Nair G, Nagaraju J. 2007. “Genetic characterization of Vibrio cholerae strains by inter simple sequence repeat-PCR” FEMS Microbiol Lett. 2007 Jul;272(2):251-8. Epub 2007 May 22
  26. N Boeckx, M W J C Jansen, C Haskovec, P Vandenberghe, V H J van der Velden and J J M van Dongen. 2005 “Identification of e19a2 BCR-ABL fusions (mu-BCR breakpoints) at the DNA level by ligation-mediated PCR” Leukemia. 2005 Jul;19(7): 1292-5
  27. Isenbarger TA, Finney M, Rios-Velazquez C, Handelsman J, Ruvkun G. 2008. “Miniprimer PCR, a new lens for viewing the microbial world.” Appl Environ Microbiol. 2008 Feb;74(3):840-9. Epub 2007 Dec 14.
  28. Calvo B, Bilbao JR, Urrutia I, Eizaguirre J, Gaztambide S, Castano L.1998. “Identification of a novel nonsense mutation and a missense substitution in the vasopressin-neurophysin II gene in two Spanish kindreds with familial neurohypophyseal diabetes insipidus” J Clin Endocrinol Metab. 1998 Mar;83(3):995-7
  29. Schiavoni G, Di Pietro M, Ronco C, De Cal M, Cazzavillan S, Rassu M, Nicoletti M, Del Piano M, Sessa R.2010 “Chlamydia pneumoniae infection as a risk factor for accelerated atherosclerosis in hemodialysis patients.” J Biol Regul Homeost  Agents. 2010 Jul-Sep; 24 (3):367-75

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